The protozoan parasite Entamoeba histolytica causes an estimated 50 million cases of invasive disease annually and is the second leading parasitic cause of death worldwide. The most common manifestations of amebic infection are colonic disease (dysentery) and liver abscesses. In addition to the lives lost there is significant morbidity associated with amebic diarrheal illness, especially in children who often suffer from malnourishment and growth retardation. E. histolytica has recently been classified as a class B agent for bioterrorism; thus development of rapid diagnostic tests, novel therapeutic agents and vaccine strategies are of even higher priority. In order to successfully develop such tests however, it is critical to understand the extent of genetic variability among clinical isolates. Our goal is to determine the extent of genetic variability among geographically distinct clinical E. histolytica isolates and to determine whether there is a correlation between parasite genotype and clinical phenotype/outcome. We will combine two powerful and complementary approaches: (i) a genomics based assay to identify genetic loci that are highly divergent or absent between the clinical isolates, and (ii) sequence analysis of >20 genetic loci to get detailed polymorphism data. Genome-wide analysis will be performed with an 11,300-clone DNA microarray that we have developed and validated for genotyping. Sequencing genetic loci under variable evolutionary pressures (introns, housekeeping genes, virulence genes, and polymorphic regions) will provide a large, diverse dataset for analysis. Our investigation will provide novel information on amebic population biology, genomic organization, and may also lead to identification of genetic loci potentially associated with virulence. This approach will also provide a number of novel genetic targets for the development of improved epidemiological markers, diagnostic assays, and treatment strategies.